1. Technical Field
The technical field of the present invention is generally the high throughput controlled nucleation of crystals, and more specifically a high-throughput method for the non-photochemical laser induced nucleation of crystals, including protein crystals.
2. Prior Art
A new method to induce and control nucleation developed by this inventor, among others, is known as non-photochemical laser induced nucleation. Garetz et al., Phys. Rev. Left. 77, 3475 (1996). In this method, short high-intensity laser pulses have been shown to induce nucleation in supersaturated solutions or urea.
The growth of crystals for structure determination relies on spontaneous nucleation. Seeded growth is normally not practical, since it would require a good quality crystal, which is not available. Other methods routinely used in small-molecule crystal growth such as cooling and the more common evaporation methods do not allow for nucleation control. Since an important purpose of crystal growth experiments is to crystallize a few large high quality crystals, uncontrolled spontaneous nucleation is a significant problem. This is true even in the case of protein crystals. For example, in the experiments using the xe2x80x9changing dropletxe2x80x9d technique, two to three nuclei per drop would usually result in large crystals of high quality while larger numbers of nuclei per drop would not. Nucleation is known to strongly depend on supersaturation. Rosenberger, F., Fundamentals of Crystal Growth (Springer-Verlag, Berlin, 1979); Chernov, A. A., Modern Crystallography III Crystal Growth (Springer-Verlag, Berlin, 1984); and McPherson, A., Eur. J. Biochem., 189, 1 (1990). Such dependence demands precise regulation of protein supersaturation, precipitant concentration, pH, purity, thermal history and temperature.
Nucleation requires that molecules aggregate together in clusters and these clusters reach a critical size where they are thermodynamically favored to grow. In addition, the molecules must overcome the entropic barrier and arrange themselves in the appropriate lattice arrangement for the resulting nuclei to be crystalline. The higher the supersaturation, the smaller the critical radius. Rosenberger F. et al., J. Crystal Growth, 168, 1 (1996). For example, because the metastable zones of protein solutions are much wider than those of small molecules, the nucleation of crystalline proteins begins at very high levels of supersaturation (often several hundred to thousand percent). Pusey, M. L., J. Crystal Growth, 110, 60 (1998); and McPherson, A., et al., Structure, 3, 759 (1995). Proteins often can nucleate and grow in an amorphous form. This generally is not desired. Recent work has shown that the second virial coefficient can be used to identify solution conditions favorable for crystallization. George, A. et al., Acta Cryst., D50, 361 (1994); Rosenbaum, D. F. et al., Phys. Rev. Lett., 76, 150 (1996); and Rosenbaum, D. F. et al., J. Crystal Growth, 169, 752 (1996).
In 1996, during a study designed to investigate whether supersaturated urea solutions would display non-linear optical properties similar to those of urea crystals because of the presence of ordered molecular clusters, the present inventor discovered serendipitously that the solutions nucleated. Garetz et al., Phys. Rev. Left. 77, 3475 (1996). The experiment involved the use of pulses of linearly polarized near infrared laser light. This wavelength of light was non-absorbing in urea solutions, which ruled out a photochemical mechanism. It was postulated that there was an alignment of molecules along the direction of the polarization due to the optical Kerr effect that reduced the entropy barrier to crystallization. Further studies of laser induced nucleation in the laboratories of the present inventor have demonstrated that the laser will induce nucleation in other substances (I-alanine, glycine, adipic acid, succinic acid), will reduce the nucleation induction time significantly when compared to an identical control.
With regard to protein crystals, complete or highly detailed steric structures of proteins are indispensable information for an understanding of the specific properties and functions of the proteins. For example, information on the three-dimensional structure of a protein can serve as the basis for understanding the mechanism of function appearance in a biochemical system by an enzyme or hormone. In many fields, such as pharmaceutical science and chemical engineering, the three-dimensional structure of a protein can provide information for basic molecular design, specific drug design, protein engineering, biochemical synthesis and the like.
X-ray crystal structural analysis is the most cogent and high-accuracy means of obtaining three-dimensional steric structural information of proteins at atomic levels at present. Thus, to determine the three-dimensional structure of a protein by X-ray crystal structural analysis, one must have protein crystals of sufficient size and quality. Crystallization of a protein currently is performed by eliminating a solvent from an aqueous or anhydrous solution containing the protein, resulting in a supersaturated state and growing a crystal. However, there are several problems in protein crystallization conducted-using the current art.
As discussed previously, it is difficult to obtain a crystal of excellent crystallinity or a large-sized single crystal. One reason may be that a biological macromolecule is readily influenced by gravity since its molecular weight is generally large and causes convection in the solution. Rosenberger, F., J. Cryst. Growth, 76, 618 (1986). This convection can reduce the crystal growth rate, or can cause anisotropic growth. Proteins also are sensitive to the crystallization conditions. The environment, pH, ionic strength and temperature of the solution, and type and dielectric constant of the buffer solution, and the like, can affect protein crystal growth. As a result, it has been difficult to obtain acceptable quantities of acceptable protein crystals, with most protein crystals being small, of less than excellent crystallinity, and in small quantities. Thus, crystallization of proteins is the weakest link in X-ray crystal structural analysis.
Others have used lasers to induce the crystallization of materials. For example, U.S. Pat. No. 4,330,363 to Biegesen et al. discloses thermal gradient control for enhanced laser-induced crystallization of predefined semiconductor areas and does not disclose or pertain to protein areas. Biegesen ""363 discloses a specific method of converting predefined areas of semiconductor material into single crystal areas and does not apply to the lased-induced nucleation of protein crystals or the controlled nucleation of protein crystals.
U.S. Pat. No. 4,737,232 to Flicstein et al. discloses a process for depositing and crystallizing a thin layer of organic material using laser energy. Flicstein ""232 discloses a specific method of depositing and crystallizing a thin layer of an organic material on a substrate, and using the laser to desorb material, and also does not apply to the lased-induced nucleation of protein crystals or the controlled nucleation of protein crystals.
U.S. Pat. No. 5,271,795 to Ataka et al. discloses a method of growing large crystals by locally controlling solution temperatures. Ataka ""795 discloses a method for growing protein crystals using the temperature dependence of solubility of a crystalline protein material, causing the protein crystals to be deposited by controlling the temperature of a localized portion of the solution. No laser is disclosed or suggested to induce or control the nucleation of protein crystals, the crystallization occurring by using warm water.
U.S. Pat. No. 5,683,935 to Miyamoto et al. discloses a method of growing semiconductor crystals only and does not disclose or pertain to protein areas. Miyamoto ""935 discloses a specific method of semiconductor crystallization by using laser light. This invention pertains to semiconductors, and does not have the same applicability to liquid solutions containing proteins.
U.S. Pat. No. 5,976,325 to Blanks discloses the laser-induced nucleation of purified aluminum hydrate crystals, including in supersaturated solutions. Although Blanks ""325 possibly can be applied to other supersaturated solution, there is no teaching or suggestion of using the process on organic materials or in fields unrelated to aluminums.
U.S. Pat. No. 6,055,106 to Grier et al. discloses a method and apparatus using laser light to assemble or direct particulate materials. Grier ""106 discloses a method for manipulating a plurality of biological objects including the crystallization of proteins. However, the invention is an optical trap that splits a single light beam into several, focuses the several light beams to form a focused spot for forming the optical trap, which is unrelated to the present invention.
High-throughput analysis systems are known in the art and are useful for various production, analysis and testing methodologies. For example, high-throughput systems in general are useful for (1) the rapid production of mixtures by automatically mixing components in series or in parallel; (2) analyzing samples by optical or analytical techniques to determine the make-up of the samples in series or in parallel; and (3) testing samples to determine the suitability of the samples for desired characteristics. High-throughput systems range from manual systems such as a series of hanging drops or pipettes acted upon sequentially, to automated systems such as two-dimensional arrays of test tubes or micro-wells acted upon by robotic systems sequentially or in parallel.
U.S. Pat. No. 5,948,363 to Gaillard illustrates a representative type of micro-well that is useful in high-throughput systems. Gaillard ""363 shows both one- and two-dimensional arrays of micro-wells. Similarly, U.S. Pat. No. 6,235,520 to Malin illustrates a high-throughput screening apparatus useful for screening the effect of test compounds. Malin ""520 shows a two-dimensional array of micro-wells in which the change of conductance can indicate various properties of the species contained in each micro-well.
U.S. Pat. No. 6,408,047 to Kitagawa discloses a method of providing high-throughput protein crystallography. Kitagawa ""047 uses a robotic arm that grasps an individual sample and is operated under automatic control, such as by a program stored within the robotic device or on a separate, connected computer. Once a sample is selected and gripped by the robotic arm, the arm is then moved to the necessary position, positioning the sample holder so that the sample will be placed in a known spatial relationship to an x-ray source and a detector. The process is repeated for subsequent samples.
PCT Patent Publication No. WO 01/51919 to Levinson discloses the use of a 96-well micro-well array for the high-throughput formation, identification, and analysis of diverse solid-forms. Levinson ""919 illustrates the use of commonly available two-dimensional arrays of micro-wells for rapidly screening many samples in parallel. Samples are placed in the micro-wells and then monitored for emerging characteristics.
Thus, while lasers have been used to induce crystallization and high-throughput systems have been used to characterize samples, it can be seen that no one has developed a successful method for the controlled high-throughput nucleation of crystal growth that results in the efficient and rapid production of crystals. Also, it can be seen that no one has developed a successful method for the start-to-finish nucleation and screening of crystals using the same high-throughput process and apparatus. Further, it can be seen that no one has developed a successful method for the controlled high-throughput nucleation of protein crystal growth that results in the efficient and rapid production of protein crystals. The present invention is directed to these needs.
The conditions required to grow many crystals are known. However, crystallization of a sufficient quantity and quality of crystals for structural analysis and other reasons is a rate-limiting step for a significant number of compounds, such as proteins. This rate-limiting step arises because even though solutions of compounds of interest generally are prepared at high levels of supersaturation, many such solutions still do not readily nucleate. In fact, it is common for some compounds to take several weeks or more to crystallize. Thus, it often is a long and arduous process to produce crystals in general and protein crystals in specific.
The present invention resolves this rate-limiting step by using non-photochemical laser-induced nucleation (NPLIN) in combination with high-throughput process methods and apparatuses to produce and screen crystals, resulting in a relatively greater number of crystals of superior quality and larger size in the same or a reduced amount of time and, potentially, with less costly human interaction. The present invention balances the desire to control crystal growth so as to achieve superior crystals with the desire to produce a larger quantity of superior crystals in less time.
In one embodiment of the present invention, one purpose of controlling protein crystal growth is to produce protein crystals of superior quality and larger size for structure determination by x-ray crystallography and other characterizing processes. For example, the general NPLIN method disclosed herein can result in an improved rate of protein crystal production and an improvement in the quality of protein crystals needed for the determination of structures. Illustrative analyses of proteins such as, for example, lysozyme and bovine insulin, and elastase shows that NPLIN results in fewer, larger protein crystals that grow in a significantly shorter amount of time than spontaneous systems with no loss of protein crystal quality. More specifically, the protein crystals themselves do not grow faster, but the time for initial nucleation is reduced, thus making the entire process faster. Therefore NPLIN offers a viable alternative for achieving high throughput (or at minimum increased throughput) crystallography.
In another embodiment of the present invention, the quantity of crystals nucleated in a solution determines ultimate size, while solution composition, pH, supersaturation, temperature and purity control the crystal quality and structural resolution. It has been found that control of nucleation at appropriate crystallization conditions would improve the size and quality of crystal. In addition, it has been found that the ability to induce nucleation on demand (or to reduce the nucleation induction time) allows more successful crystal growth in shorter time periods.
NPLIN uses short high-intensity laser pulses to induce nucleation in supersaturated solutions, including supersaturated protein solutions. The laser induces nucleation only in the area where the beam is focused or passes through, resulting in much fewer nuclei than would be achieved by spontaneous nucleation. In addition, the laser reduces nucleation time significantly. By controlling the number of nuclei, larger crystals result, which gives better results in x-ray structure analysis. One benefit of the invention is an improved rate of production and quality of crystals needed for determination of structures. Another benefit of the invention is a non-intrusive method for nucleating and growing crystals in that no seeds or other nucleating matter is introduced to the supersaturated solution, resulting in a cleaner and higher quality crystal.
NPLIN coupled with high-throughputmethodologies and apparatuses further improves the rate at which such higher quality and larger crystals can be formed. As discussed above, crystals often from and grow in a relatively slow process. By using high-throughput techniques, multiple crystals can be nucleated by NPLIN in sequence or in parallel, and grown contemporaneously, resulting in a higher yield of higher quality and/or larger crystals.
For example, the present method, termed high-throughput non-photochemical laser-induced nucleation (HTNPLIN), can be used for increased throughput crystallization using, for example, an array of hanging or sitting drops of supersaturated solutions containing the compounds of interest. Further, HTNPLIN can be applied to various alternative reaction vessels including, but not limited to, cuvettes, pipettes, tubes and micro-wells. Additionally, the laser can be pulsed through an aligned series or array of samples simultaneously and the beam can be split to allow the laser to be pulsed through multiple arrays at once. In addition, the arrays can be moved with time as the laser pulses to allow a large number of the samples to be exposed to the laser. HTNPLIN also can be automated to allow more efficient and more consistent laser exposure.
The method of non-photochemical laser induced nucleation of crystals as disclosed below, when combined with standard methods of crystal growth, results in fewer larger crystals of better quality. However, when further combined with high-throughput methodologies and apparatuses, more of the larger crystals of better quality can be produced in the same or less time. In addition, this method allows a reduction in the nucleation induction time so as to increase the overall rate of crystal growth. Thus, this results in an improvement in the quality and size of crystals and allows for more successful experiments per unit time. Further, the reduction of the number of crystals nucleating and growing in any given volume results in a cleaner, more efficient process.
By employing the present invention, those skilled in the art can identify and optimize appropriate conditions of power, pulse length and polarization for the laser-induced nucleation of a number of different compounds so as to provide larger and higher diffraction quality crystals compared to current methods at identical conditions. Further, the present invention results in a reduction of the nucleation induction time needed for crystals when compared with spontaneous nucleation at identical conditions. Also, this is the first time that laser-induced nucleation has been combined with high-throughput methodologies to initiate the formation of and grow multiple crystals in supersaturated solutions that will not spontaneously nucleate to form crystals.
The advantages of the present invention will become apparent to those of ordinary skill in the art when the following detailed description of the preferred embodiments is read in conjunction with the appended figure. The present invention is directed to these needs and advantages.